DESCRIPTION: Successes in isolating human broadly neutralizing antibodies (bnAbs) and mapping the isolated monoclonal antibody (mAb) epitopes have helped to reveal several sites of vulnerability on the HIV-1 envelope glycoprotein (Env); however, the isolated bnAbs may only account for part of the donor sera neutralizing activity, indicating unknown targets on HIV-1 Env. Additionally, most of the isolated bnAbs have demonstrated unusual coding sequences implicating potential elicitation barriers. To progress on our quest to elicit protective antibodie through vaccination, we propose to identify and characterize new functional antibodies that may be more feasible for elicitation. Using a resurfaced gp120 stabilized core protein RSC3 as a probe, we previously identified the bnAb VRC01 and its class of mAbs that target the CD4-binding site (CD4bs) of gp120. By the same probing method, we recently identified a new mAb, VRC-PG05, from an infected individual who also developed the VRC01-class bnAb VRC-PG04. To date VRC-PG05 is the least somatically mutated neutralizing mAb against HIV-1 and its coding sequences do not seem uncommon in the human antibody repertoire. Atomic-level structure of VRC-PG05 in complex with gp120 revealed a novel glycopeptide epitope centering on two highly conserved glycans at N262 and N448 (based on HXB2 numbering) on gp120. Removal of either glycosylation site resulted in complete loss of VRC-PG05 activity, confirming the importance of the N262/N448 glycans for the mAb recognition. Based on the novelty of the glycopeptide epitope, the ability for co-elicitation with the CD4bs-directed functional mAbs, the relatively normal coding sequences, as well as the virus neutralization function, we consider VRC-PG05 and its epitope an attractive immune target and thus propose to generalize its findings to additional infected individuals. We hypothesize that 1) the newly discovered N262/N448 glycan site on gp120 is immunogenic, and antibodies targeting this site are frequently elicited in HIV-1-infected individuals; and 2) genetic sequences that encode functional antibodies targeting the N262/N448 glycan site do not require unusual features shared by most of the identified HIV-1 bnAbs. We have derived recombinant gp120 proteins with point mutations to detect the N262/N448 specificity by ELISA. We applied this monomeric gp120 ELISA to screen plasma samples from 85 HIV-1 seroconverters, and so far identified 17 (20%) who possessed antibodies with the N262/N448 specificity. These preliminary results support our first hypothesis regarding the immunogenicity of the newly discovered N262/N448 glycan site. From individuals with this antibody specificity, we propose to isolate the corresponding mAbs, determine the mAb anti-viral functions, and examine if the mAb coding sequences display any unusual features shared by most of the identified bnAbs. If successful, this project will yield new functional anti-HIV-1 mAbs that may be more feasible for elicitation through vaccination.